SCREENING METHOD FOR PROKINETIC AGENT

Info

Publication number: 20100129345
Type: Application
Filed: Oct 5, 2007
Publication Date: May 27, 2010
Patent Grant number: 8034574
Applicant: Astellas Pharma Inc. (Tokyo)
Inventors: Katsura Nozawa (Tokyo), Eri Shoda (Tokyo), Hitoshi Doihara (Tokyo), Ryosuke Kojima (Tokyo)
Application Number: 12/302,808

Abstract

The present invention provides a screening tool and screening method for obtaining a substance useful as a prophylactic and/or therapeutic drug for diseases associated 5 with 5-HT production/secretion abnormalities, including digestive organ diseases, and a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, including digestive organ diseases. Examples of digestive organ diseases in the present invention include constipation type irritable bowel syndrome, functional dyspepsia, constipation, diarrhea type irritable bowel syndrome, diarrhea and vomiting.

Description

Description

TECHNICAL FIELD

The present invention relates to a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, including digestive organ diseases, and a screening method and tool therefor and the like.

BACKGROUND ART

Serotonin (hereinafter referred to as 5-HT) is abundantly contained in fruits such as bananas, vegetables, harmful plants and the like. In animals, 90% of 5-HT in the living body is present in the gastrointestinal tract. The 5-HT in the gastrointestinal tract is biosynthesized in gastrointestinal mucosal chromaffin cells (enterochromaffin cells; hereinafter referred to as EC cells), entering the blood circulation, and is transported to the whole body. The 5-HT released upon chemical stimulation or mechanical stimulation of the gut binds to 5-HT receptors of target cells to cause physiological reactions. As 5-HT receptors involved in digestive tract movement functions, 5-HT receptor 1, 5-HT receptor 2, 5-HT receptor 3, 5-HT receptor 4, 5-HT receptor 7 and the like have been recognized. These receptors have been shown to be expressed in the nerve cells and smooth muscles of the gastrointestinal tract. The 5-HT released from EC cells controls digestive tract movement functions via the nerve cells and smooth muscles that express these 5-HT receptors. Hence, 5-HT is thought to be a kind of hormone that regulates gastrointestinal tract functions (non-patent document 1).

Although it has been known that when chemical stimulation or mechanical stimulation is given to EC cells, 5-HT release is promoted and gut movement is accentuated, little has been demonstrated about what is the molecular mechanism by which the above-described promotion of 5-HT release from EC cells is caused.

Currently, in the clinical settings for the field of digestive organ diseases, drugs that control the activity of 5-HT receptors are used. For example, 5-HT receptor 3 inhibitors are used to treat diarrhea type IBS, as antiemetics, and for other purposes, whereas 5-HT receptor 4 activators are used to treat constipation type IBS and digestive organ dysfunction and for other purposes. Because many IBS patients have an abnormality in postprandial blood 5-HT level, it has been demonstrated that 5-HT is associated with the pathologic condition. However, not many therapeutic drugs offer high satisfaction for patients with constipation type IBS or other digestive organ diseases (non-patent document 1).

TRPA1, belonging to the TRP (Transient Receptor Potential) channel family, was recently reported to be a temperature-sensitive channel, and TRPA1 was reported to become activated at temperatures of 17° C. or lower, and to become activated by nociceptive cold stimulation (non-patent document 2). It was also demonstrated that TRPA1 serves as a ligand agonizing ion channel that becomes activated not only by low temperatures, but also by stimulants such as mustard (non-patent document 3, patent document 1).

Furthermore, from experiments using TRPA1-deficient mice, it was demonstrated that TRPA1 activates primary afferent nociceptors to cause inflammatory algesia (non-patent document 4). From these experimental results, it is thought that TRPA1 plays an important part in the transmission mechanism by which an exogenous stimulant and an endogenous pain inducer cause inflammatory pain.

Although functions of TRPA1 concerning the sensory nerves and the like are already commonly known as described above, there is no research report on the roles of TRPA1 in the digestive tract, and its functions in the gut remain elusive.

[Patent document 1] WO2005/089206

[Non-patent document 1] TEXTBOOK of Gastroenterolorogy, Fourth Edition, ISBN 0-7817-2861-4

[Non-patent document 2] Cell, Vol. 112, 819-829(2003)

[Non-patent document 3] Nature, Vol. 427, 260-265(2004)

[Non-patent document 4] Cell, Vol. 124, 1269-1282(2006)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

It is an object of the present invention to provide a screening tool and screening method for obtaining a substance useful as a prophylactic/therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, including digestive organ diseases, and a means that can be used for the above-described screening, and to further provide a novel prophylactic/therapeutic drug for digestive organ diseases, and/or diseases associated with 5-HT production/secretion abnormalities, and the like.

Means of Solving the Problems

The present inventors found that TRPA1 is expressed in 5-HT releasing cells responsible for 5-HT production/secretion to regulate the 5-HT release from these cells, in more detail, the 5-HT release from such cells is specifically promoted by activation of TRPA1, and that the 5-HT release from such cells is specifically suppressed by inhibition of TRPA1. Based on these findings, the present inventors conceptualized that a substance capable of regulating the expression or channel activity of TRPA1 can be useful as a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities (e.g., digestive organ diseases), and that a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities can be obtained by screening for a substance capable of regulating the expression or channel activity of TRPA1, and developed the present invention.

Accordingly, the present invention provides the following:

[1] A screening method for a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, comprising evaluating a test substance to determine whether or not the test substance is capable of regulating the expression or channel activity of TRPA1.
[2] A screening method for a prophylactic and/or therapeutic drug for digestive organ diseases, comprising evaluating a test substance to determine whether or not the test substance is capable of regulating the expression or channel activity of TRPA1.
[3] The screening method according to [1] or [2] above, comprising the following steps (a) to (c):
(a) a step for bringing a test substance into contact with mammalian cells that are expressing TRPA1;
(b) a step for analyzing the expression or channel activity of TRPA1; and
(c) a step for selecting a substance capable of regulating the expression or channel activity of TRPA1.
[4] The screening method according to [3] above, wherein the mammalian cells that are expressing TRPA1 are chromaffin cells, pancreatic β cells or cells transformed with a TRPA1 expression vector.
[5] The screening method according to any one of [1] to [4] above, wherein the screening method is performed using a TRPA1 activator or a TRPA1 inhibitor.
[6] The screening method according to any one of [1] to [5] above, wherein the regulation of the expression or channel activity of TRPA1 is promotion of the expression or channel activity of TRPA1.
[7] The screening method according to any one of [1] to [5] above, wherein the regulation of the expression or channel activity of TRPA1 is suppression of the expression or channel activity of TRPA1.
[8] The screening method according to [3] above, which is a method of screening for a prophylactic or therapeutic drug for constipation type irritable bowel syndrome, functional dyspepsia or constipation by selecting a substance capable of promoting the expression or channel activity of TRPA1.
[9] The screening method according to [3] above, which is a method of screening for a prophylactic or therapeutic drug for diarrhea type irritable bowel syndrome, diarrhea or vomiting is by selecting a substance capable of suppressing the expression or channel activity of TRPA1.
[10] A screening tool for a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, comprising cells transformed with a TRPA1 expression vector.
[11] A screening tool for a prophylactic and/or therapeutic drug for a digestive organ disease, comprising cells transformed with a TRPA1 expression vector.
[12] The screening tool according to [11] above, wherein the digestive organ disease is irritable bowel syndrome, functional dyspepsia, constipation, diarrhea or vomiting.
[13] A prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, comprising a substance capable of regulating the expression or channel activity of TRPA1.
[14] A prophylactic and/or therapeutic drug for a digestive organ disease, comprising a substance capable of regulating the expression or channel activity of TRPA1.
[15] A method of producing a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, comprising screening for a substance capable of regulating the expression or channel activity of TRPA1, and preparing the substance obtained by the screening as a pharmaceutical preparation.
[16] The production method according to [15] above, comprising the following steps (a) to (d):
(a) a step for bringing a test substance into contact with mammalian cells that are expressing TRPA1;
(b) a step for analyzing the expression or channel activity of TRPA1;
(c) a step for selecting a substance capable of regulating the expression or channel activity of TRPA1; and
(d) a step for preparing the substance obtained in the step (c) as a pharmaceutical preparation.
[17] A prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, comprising a substance that can be obtained by the screening method according to [1] or [2] above.
[18] A prophylactic and/or therapeutic method for diseases associated with 5-HT production/secretion abnormalities, comprising administering a substance that can be obtained by the screening method according to [1] or [2] above to a patient in need of prevention and/or treatment.
[19] A use of a substance that can be obtained by the screening method according to [1] or [2] above, in producing a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities.
[20] A screening method for a substance that exhibits a specified pharmacological effect, and that does not have the capability of regulating 5-HT release, comprising evaluating a test substance to determine whether or not the test substance that exhibits a specified pharmacological effect is capable of regulating the expression or channel activity of TRPA1.
[21] The screening method according to [20] above, comprising the following steps (a) to (c):
(a) a step for bringing a test substance that exhibits a specified pharmacological effect into contact with mammalian cells that are expressing TRPA1,
(b) a step for analyzing the expression or channel activity of TRPA1; and
(c) a step for selecting a substance that exhibits a specified pharmacological effect, and that does not regulate the expression or channel activity of TRPA1.
[22] A method of producing a pharmaceutical, comprising screening for a substance that exhibits a specified pharmacological effect, and that does not regulate the expression or channel activity of TRPA1, and preparing the substance obtained by the screening as a pharmaceutical preparation.
[23] A use of TRPA1 as a screening tool for a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities.
[24] A use of TRPA1 as a screening tool for a prophylactic and/or therapeutic drug for a digestive organ disease.

Effect of the Invention

The screening tool and screening method of the present invention can be useful in, for example, developing a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, including digestive organ diseases, and a pharmaceutical that exhibits a specified pharmacological effect, and that is not desired to act as a result of the capability of regulating 5-HT release (e.g., adverse reactions in digestive organs).

The pharmaceutical of the present invention can be useful as, for example, a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, including digestive organ diseases, and as a pharmaceutical that exhibits a specified pharmacological effect, and that is not desired to act as a result of the capability of regulating 5-HT release. The present invention also provides a method of producing such a pharmaceutical.

BRIEF DESCRIPTION OF THE DRAWINGS

[FIG. 1] shows the results of an examination of changes in intracellular Ca²⁺ concentration with the addition of 90 μM of each of allyl isothiocyanate (A), cinnamic aldehyde (B), and acrolein (C). The axis of ordinates indicates fluorescence intensity in terms of intracellular Ca²⁺ concentration; the axis of abscissas indicates the time course after addition of each sample. □ shows the results for TRPA1-expressing CHO-K1 cells; ∘ shows the results for control CHO-K1 cells.

[FIG. 2] shows distributions of TRPA1 mRNA expression in various human tissues. The values shown are relative to the expression level of the human G3PDH gene as 100%.

[FIG. 3] shows the results of in situ hybridization staining of the TRPA1 gene using the human duodenum. In an investigation using an antisense probe, intense color development was found in some cells in the epithelium of the human duodenum (left panel; arrow). On the other hand, in an investigation using a sense probe, no staining was observed (right panel).

[FIG. 4] shows the results obtained by performing in situ hybridization staining of the TRPA1 gene using the human duodenum, and then immunologically staining the same section with an anti-serotonin antibody. Cells that concurrently expressed TRPA1 and serotonin are shown by the arrow.

[FIG. 5] shows the results of measurements of the amount of serotonin released when each of allyl isothiocyanate, cinnamic aldehyde, and acrolein was added to RIN14B cells. Each of them concentration-dependently promoted the release of serotonin from the RIN14B cells.

[FIG. 6] shows the results of measurements of the amount of serotonin released when an siRNA of TRPA1 was introduced to RIN14B cells. #971 siRNA of TRPA1 suppressed cinnamic aldehyde-induced serotonin release from the RIN14B cells (mean±SD).

[FIG. 7] shows the results of measurements of the amount of serotonin released when EC cells purified from the rat small intestine were treated with allyl isothiocyanate and cinnamic aldehyde. Both allyl isothiocyanate and cinnamic aldehyde promoted the release of serotonin from the EC cells.

[FIG. 8-1] shows the results of a measurement of dog digestive tract movement by the strain gauge force transducer method. Allyl isothiocyanate accentuated stomach movement just after administration (solid arrow) and induced GMC (outlined arrow). The timing of administration is shown by the broken line.

[FIG. 8-2] shows the results of a measurement of dog digestive tract movement by the strain gauge force transducer method. The vehicle suppressed stomach movement just after administration (solid arrow), and did not induce GMC (outlined arrow). The timing of administration is shown by the broken line.

[FIG. 9] shows the results of a measurement of the action of allyl isothiocyanate in an experiment to measure digestive tract water secretion. Allyl isothiocyanate concentration-dependently accentuated water secretion compared to the vehicle control. (N=6, mean±SE)

*: p<0.01 vs saline group (Dunnett's test)

[FIG. 10] shows the results of a measurement of the action of allyl isothiocyanate in a loperamide-induced constipation model. Allyl isothiocyanate concentration-dependently shortened bead discharge time compared to the vehicle control. (N=6, mean±SE)

#: p<0.05 vs control (Student's t-test), *: P<0.05 vs vehicle (Dunnett's test)

BEST MODE FOR CARRYING OUT THE INVENTION 1. Screening Tools

The present invention provides screening tools for a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, including digestive organ diseases. Examples of the screening tools of the present invention include polypeptide type screening tools and cell type screening tools.

(1) Polypeptide Type Screening Tool

Examples of polypeptides that can be used as the screening tool of the present invention include the following (i) to (iii):

(i) a polypeptide consisting of the same amino acid sequence as mammalian TRPA1;
(ii) (a) a polypeptide that comprises an amino acid sequence of mammalian TRPA1, and that becomes activated by a TRPA1 activator to exhibit cation-transmitting ion channel activity, or (b) a polypeptide that comprises an amino acid sequence of mammalian TRPA1 wherein 1 to 10 amino acids have been deleted, substituted, and/or inserted, and that becomes activated by a TRPA1 activator to exhibit cation transmitting ion channel activity [the tool polypeptide consisting of polypeptide (a) and the tool polypeptide consisting of polypeptide (b) are hereinafter together referred to as functionally equivalently modified tool polypeptides]; or
(iii) a polypeptide that consists of an amino acid sequence having an identity of 80% or more to an amino acid sequence of mammalian TRPA1, and that becomes activated by a TRPA1 activator to exhibit cation transmitting ion channel activity (hereinafter referred to as an identical tool polypeptide).

Hereinafter, these various polypeptides that can be used as the polypeptide type screening tool of the present invention are generically referred to as screening tool polypeptide or TRPA1 (polypeptide).

As used herein, “becomes activated by a TRPA1 activator to exhibit cation transmitting ion channel activity” means that when the current response value or calcium inflow or inflow of another cation for cells having a subject polypeptide expressed forcibly therein, or cells naturally expressing the same, stimulated with a TRPA1 activator, is compared with that for non-stimulated cells, the current response value or calcium inflow or inflow of the other cation for the stimulated cells is higher than the current response value or calcium inflow or inflow of the other cation for the non-stimulated cells. For example, a comparison of calcium inflow can be confirmed by the method described in Example 4, 5 or 13. Regarding the extent of the increase in calcium inflow, the P value is preferably not more than 0.05, and the P value is more preferably not more than 0.01, when a test is performed to determine the significant difference from the calcium inflow for non-stimulated cells.

The screening tool polypeptide of the present invention more preferably also exhibits cesium, sodium, and magnesium ion transmitting ion channel activity.

Examples of each polypeptide consisting of an amino acid sequence of mammalian TRPA1, which can be used as the polypeptide type screening tool of the present invention, include human-, mouse-, and rat-derived TRPA1 (e.g., SEQ ID NO:2, 4, and 6, respectively). TRPA1 has an amino acid sequence identity of 79.7% and a nucleotide identity of 80.7% between humans and mice, an amino acid sequence identity of 79.6% and a nucleotide identity of 79.9% between humans and rats, and an amino acid sequence identity of 96.6% and a nucleotide identity of 94.3% between mice and rats. In the present invention, human-derived TRPA1 (e.g., SEQ ID NO:2) is particularly preferred.

Although information on the amino acid sequence of TRPA1 and ligands that activate TRPA1 is available from various literature documents, none of them discloses or suggests the involvement in 5-HT release from EC cells or digestive tract movement.

Preferred as a functionally equivalently modified tool polypeptide that can be used as the polypeptide type screening tool of the present invention is (a) a polypeptide that consists of an amino acid sequence of mammalian TRPA1 wherein a total of 1 to 10 (more preferably 1 to 7, still more preferably 1 to 5, particularly preferably 1 or 2) amino acids have been deleted, substituted, inserted, and/or added at one or a plurality of sites, and that becomes activated by a TRPA1 activator to exhibit cation transmitting ion channel activity, is or (b) a polypeptide that comprises an amino acid sequence of mammalian TRPA1, and that becomes activated by a TRPA1 activator to exhibit cation transmitting ion channel activity.

Examples of a polypeptide that comprises an amino acid sequence of mammalian TRPA1, and that becomes activated by a TRPA1 activator to exhibit cation transmitting ion channel activity also include a polypeptide consisting of an amino acid sequence of mammalian TRPA1 wherein an appropriate marker sequence and the like have been added to the N terminus and/or C terminus thereof (that is, a fusion polypeptide), as far as it becomes activated by a TRPA1 activator to exhibit calcium ion transmitting ion channel activity.

As the aforementioned marker sequence, for example, a sequence for facilitating the confirmation of the expression, confirmation of the intracellular localization, or purification and the like of the polypeptide can be used; examples include FLAG epitope, hexa-histidine/tag, hemagglutinin/tag, or myc epitope and the like.

An identical tool polypeptide that can be used as the polypeptide type screening tool of the present invention has an identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, to an amino acid sequence of mammalian TRPA1, and one having an identity of 99% or more is particularly preferable. Herein, the degree of the aforementioned “identity” is determined by the ClustalV method using MegAlign (DNASTAR).

(2) Cell Type Screening Tool

Cells that can be used as the cell type screening tool of the present invention (hereinafter referred to as screening tool cells) are not particularly limited, as far as they express the aforementioned screening tool polypeptide when used as a cell type screening tool; they can be transformant cells forcibly expressing the aforementioned polypeptide by transformation with a foreign gene, or they can be natural cells expressing a screening tool polypeptide or a cell line thereof (e.g., RIN14B cells). Screening tool cells can also be provided in the form of a tissue containing the cells.

As screening tool cells that can be used as the cell type screening tool of the present invention, transformant cells incorporating the TRPA1 gene are preferred. Examples of such cells include the following (i) to (iii):

(i) transformant cells expressing a polypeptide consisting of an amino acid sequence of mammalian TRPA1;
(ii) transformant cells expressing a functionally equivalently modified tool polypeptide; or
(iii) transformant cells expressing an identical tool polypeptide.

Preferably, the mammalian cells expressing TRPA1 can be 5-HT releasing cells. As used herein, “5-HT releasing cells” refer to cells capable of releasing 5-HT through a mechanism of control mediated by TRPA1; examples include EC cells (e.g., EC cells derived from tissues such as gastrointestinal tract mucosa, lungs, skin, and pancreas) and endocrine cells such as pancreatic β cells. Such cells include normal cells and cancer cells.

The screening tools (1) and (2) above in the present invention can be useful in screening for a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, including digestive organ diseases. Such diseases include digestive organ diseases and non-digestive organ diseases. Examples of digestive organ diseases include irritable bowel syndrome (e.g., constipation type irritable bowel syndrome, diarrhea type irritable bowel is syndrome), functional dyspepsia, constipation, diarrhea, and vomiting. Examples of non-digestive organ diseases include eating disorders (e.g., bulimia, anorexia nervosa), pain, migraine, sleep disturbance (e.g., insomnia), mental disorders (e.g., depression, anxiety disorders, schizophrenia), blood coagulation disorders (e.g., platelet aggregation dysfunction, thrombosis, pulmonary thromboembolism), and carcinoid tumor.

Production of the screening tool of the present invention can be performed in accordance with a commonly known method (for example, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1989, WO02/052000, or WO02/053730).

The method of producing a polynucleotide that encodes the screening tool polypeptide of the present invention (hereinafter referred to as a screening tool polynucleotide) is not particularly limited; examples include (a) the method based on a polymerase chain reaction (PCR), (b) the method based on a conventional method of gene engineering technology (that is, a method wherein a transformant strain containing a desired cDNA is selected from among transformant strains resulting from transformation with a cDNA library), or (c) the chemical synthesis method and the like. These methods of production are hereinafter described in sequence.

In the PCR-based method [the aforementioned method of production (a)], for example, by the procedures shown below, the screening tool polynucleotide of the present invention can be produced.

That is, mRNA is extracted from cells (for example, human, mouse, or rat cells) or tissue having the capability of producing the screening tool polypeptide of the present invention. Next, on the basis of the base sequence of a polynucleotide that encodes the aforementioned polypeptide, a set of two primers between which full-length mRNA corresponding to the aforementioned polypeptide can be sandwiched, or a set of two primers between which the mRNA region of a portion thereof can be sandwiched, is prepared. By performing a reverse transcriptase-polymerase chain reaction (RT-PCR) while adjusting reaction conditions (for example, denaturation temperature or denaturant addition conditions and the like) as appropriate, a full-length cDNA that encodes the screening tool polypeptide of the present invention or a portion thereof can be obtained.

By performing PCR with a cDNA prepared from mRNA prepared from cells (for example, human, mouse, or rat cells) or tissue having the capability of producing the aforementioned polypeptide using a reverse transcriptase, or a commercially available cDNA derived from human, mouse, or rat cells or tissue, as the template, a full-length cDNA that encodes the aforementioned polypeptide or a portion thereof can also be obtained.

The thus-obtained full-length cDNA or portion thereof may be integrated into an appropriate expression vector and expressed in host cells, whereby the aforementioned polypeptide can be produced.

In the method based on a conventional method of gene engineering technology [the aforementioned method of production (b)], for example, the screening tool polynucleotide of the present invention can be produced per the procedures shown below.

First, with mRNA prepared by the aforementioned PCR-based method as the template, using a reverse transcriptase, a single-stranded cDNA is synthesized, after which a double-stranded cDNA is synthesized from this single-stranded cDNA.

Next, a recombination plasmid harboring the aforementioned double-stranded cDNA is prepared, after which it is introduced to Escherichia coli (for example, DH5α strain, HB101 strain, or JM109 strain) to transform the strain, and a recombinant is selected with, for example, drug resistance to tetracycline, ampicillin, or kanamycin as the index. When the host cell is Escherichia coli, transformation of the host cell can be performed by Hanahan's method (Hanahan, D. J., Mol. Biol., 166, 557-580, 1983). Commercially available competent cells can also be used. In addition to plasmids, phage vectors such as the lambda series can also be used as vectors.

As a method of selecting a transformant strain having a desired cDNA from among the transformant strains thus obtained, for example, (1) a screening method based on hybridization using a synthetic oligonucleotide probe, or (2) a screening method based on hybridization using a PCR-prepared probe can be adopted.

A method of collecting the screening tool polynucleotide of the present invention from the desired transformant strain obtained can be performed in accordance with a commonly known method. For example, this method can be performed by separating a fraction corresponding to the plasmid DNA from the cells, and cleaving out the cDNA region from the plasmid DNA obtained.

In the method based on chemical synthesis [the aforementioned method of production (c)], by, for example, binding a DNA fragment produced by chemical synthesis, the screening tool polynucleotide of the present invention can be produced. Each DNA can be synthesized using a DNA synthesizer [for example, Oligo 1000M DNA Synthesizer (produced by Beckman), or 394 DNA/RNA Synthesizer (produced by Applied Biosystems) and the like].

Sequencing of the DNAs obtained by the various methods described above can be performed by, for example, the Maxam-Gilbert method of chemical modification (Maxam, A. M. and Gilbert, W., Methods in Enzymology, 65, 499-559, 1980), the dideoxynucleotide chain termination method (Messing, J. and Vieira, J., Gene, 19, 269-276, 1982) and the like.

By again integrating the isolated screening tool polynucleotide of the present invention into an appropriate vector to transform host cells (including eukaryotic host cells and prokaryotic host cells), the cells or screening tool cells of the present invention can be obtained. It is also possible to express the polynucleotide in the respective host cells by introducing an appropriate promoter and a sequence involved in the character expression into these vectors.

For example, eukaryotic host cells include cells of vertebral animals, insects, yeast and the like; examples of vertebral animal cells include COS cells, which are monkey cells (Gluzman, Y., Cell, 23, 175-182, 1981), the dihydrofolate reductase-deficient line of Chinese hamster ovarian cells (CHO) (Urlaub, G. and Chasin, L. A., Proc. Natl. Acad. Sci. USA, 77, 4216-4220, 1980), fetal human kidney-derived HEK293 cells, 293-EBNA cells (Invitrogen) prepared by introducing the Epstein-Barr virus EBNA-1 gene to the aforementioned HEK293 cells, and the like.

As an expression vector for vertebral animal cells, one having a promoter lying upstream of the polynucleotide to be expressed, an RNA splicing site, a polyadenylation site, and a transcription termination sequence and the like can usually be used, and can have a replication origin as required. Examples of the aforementioned expression vector include, for example, pSV2dhfr, which has the early promoter of SV40 (Subramani, S. et al., Mol. Cell. Biol., 1, 854-864, 1981), pEF-BOS, which has a human elongation factor promoter (Mizushima, S. and Nagata, S., Nucleic Acids Res., 18, 5322, 1990), or pCEP4, which has a cytomegalovirus promoter (Invitrogen), and the like.

When COS cells are used as the host cells, an expression vector that has an SV40 replication origin, that is capable of self-proliferating in the COS cells, and that is further provided with a transcription promoter, a transcription termination signal, and an RNA splicing site, can be used; examples include pME18S (Maruyama, K. and Takebe, Y., Med. Immunol., 20, 27-32, 1990), pEF-BOS (Mizushima, S. and Nagata, S., Nucleic Acids Res., 18, 5322, 1990), or pCDM8 (Seed, B., Nature, 329, 840-842, 1987) and the like.

The aforementioned expression vector can be incorporated into COS cells by, for example, the DEAE-dextran method (Luthman, H. and Magnusson, G., Nucleic Acids Res., 11, 1295-1308, 1983), the calcium phosphate-DNA co-precipitation method (Graham, F. L. and van der Ed, A. J., Virology, 52, 456-457, 1973), a method using a commercially available transfection reagent (for example, FuGENE™ 6 Transfection Reagent; produced by Roche Diagnostics), or electroporation (Neumann, E. et al., EMBO J., 1, 841-845, 1982) and the like.

When CHO cells are used as the host cells, transformant cells that stably produce the screening tool polypeptide of the present invention can be obtained by co-transfecting an expression vector harboring a polynucleotide for the screening tool of the present invention with a vector capable of expressing the neo gene, which functions as a G418 resistance marker, for example, pRSVneo (Sambrook, J. et al. Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1989) or pSV2-neo (Southern, P. J. and Berg, P., J. Mol. Appl. Genet., 1, 327-341, 1982) and the like, and selecting a G418-resistant colony.

When 293-EBNA cells are used as the host cells, pCEP4 (Invitrogen), which has the replication origin of Epstein-Barr virus, and which is capable of self-replication in 293-EBNA cells, and the like can be used as the expression vectors.

The transformants can be cultured in accordance with a conventional method; by the aforementioned cultivation, the screening tool polypeptide of the present invention is produced through the cell membrane. As media that can be used for the aforementioned cultivation, various media in common use can be selected as appropriate according to the host cells adopted. For example, in the case of COS cells, for example, a medium prepared by adding as required a serum component such as fetal bovine serum (FBS) to a medium such as RPMI-1640 medium or Dulbecco's modified Eagle medium (DMEM) can be used. In the case of 293-EBNA cells, a medium prepared by adding G418 to a medium such as Dulbecco's modified Eagle medium (DMEM) supplemented with a serum component such as fetal bovine serum (FBS) can be used.

The screening tool polypeptide of the present invention, produced by culturing the transformant, can be separated and purified by various commonly known methods of separation based on a physical property, biochemical property or the like of the aforementioned polypeptide. Specifically, cells or a cell membrane fraction containing the aforementioned polypeptide can be subjected to, for example, treatment with an ordinary protein precipitant, ultrafiltration, various liquid chromatographies [for example, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, or high performance liquid chromatography (HPLC) and the like], or dialysis, or a combination thereof and the like, to purify the aforementioned polypeptide.

By fusing the screening tool polypeptide of the present invention with a marker sequence in frame, confirmation of the expression or purification and the like of the aforementioned polypeptide is facilitated. Examples of the aforementioned marker sequence include FLAG epitope, hexa-histidine/tag, hemagglutinin/tag, or myc epitope and the like. By inserting a specific amino acid sequence recognized by a protease (for example, enterokinase, factor Xa, or thrombin and the like) between the marker sequence and the aforementioned polypeptide, it is possible to cleave out the marker sequence portion with these proteases.

2. Screening Methods

The present invention provides screening methods comprising evaluating a test substance to determine whether or not the test substance is capable of regulating the expression or channel activity of TRPA1.

The screening methods of the present invention can be roughly divided into a screening method for a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, including digestive organ diseases, comprising selecting a substance capable of regulating the expression or channel activity of TRPA1 (screening method I), and a screening method for a substance not having the capability of regulating 5-HT release, comprising selecting a substance that does not regulate the expression or channel activity of TRPA1 (screening method II).

Hereinafter, the individual screening methods are described in detail.

2.1. Screening Method Comprising Selecting a Substance Capable of Regulating the Expression or Channel Activity of TRPA1

The present invention provides a screening method comprising evaluating a test substance to determine whether or not the test substance is capable of regulating the expression or channel activity of TRPA1, and selecting a substance capable of regulating the expression or channel activity of TRPA1 (screening method I).

The test substance subjected to the screening method I is not particularly limited; for example, various commonly known compounds (including peptides) registered with chemical files, a set of compounds obtained by combinatorial chemistry technology (Terrett, N. K. et al. Tetrahedron, 51, 8135-8137, 1995), or a set of random peptides prepared by applying the phage display method (Felici, F. et al., J. Mol. Biol., 222, 301-310, 1991) and the like can be used. Natural components derived from microorganisms, plants, marine organisms or animals (for example, culture supernatant or tissue extract) and the like can also be used as test substances for screening. Furthermore, compounds (including peptides) selected by the screening method of the present invention, for example, compounds (including peptides) prepared by chemically or biologically modifying allyl isothiocyanate, cinnamic aldehyde, or acrolein, can be used.

In detail, the screening method I of the present invention comprises the following steps (a) to (c):

(a) a step for bringing a test substance into contact with the screening tool of the present invention (e.g., screening tool cells);
(b) a step for analyzing (measuring, detecting) the expression or channel activity of TRPA1; and
(c) a step for selecting a substance capable of promoting or suppressing the expression or channel activity of TRPA1.

The expression of TRPA1 can be analyzed by, for example, using the method described below in mammalian cells expressing TRPA1 (that is, screening tool cells).

The expression of TRPA1 can also be analyzed using cells that allow a reporter assay for a TRPA1 transcription regulatory region. The cells that allow an reporter assay for the TRPA1 transcription regulatory region can be cells transformed with an expression vector harboring a TRPA1 transcription regulatory region and a reporter gene functionally joined to the region. The TRPA1 transcription regulatory region is not particularly limited, as far as it is a region capable of controlling the expression of TRPA1; examples include a region up to about 2 kbp upstream of the transcription initiation point, or a region that consists of the base sequence of the region wherein one or more bases have been deleted, substituted or added, and that has the capability of controlling the transcription of a target gene and the like. Examples of reporter genes include the GFP (green fluorescent protein) gene, the GUS (β-glucuronidase) gene, the LUC (luciferase) gene, the CAT (chloramphenicol acetyltransferase) gene and the like.

As used herein, “a substance that promotes the activity of the channel” has the same definition as that for “a substance that activates the channel” to refer to a substance that activates the ion channel by being brought into contact with a test substance, including both a substance that directly activates the channel, like TRPA1 activators, and a substance that promotes the activation of a substance that directly activates the channel. By performing the above-described step in the presence of a TRPA1 activator, a substance that promotes the activation of TRPA1 by a TRPA1 activator can be screened for; a screening method for a substance that promotes the activation of the aforementioned polypeptide by a TRPA1 activator is also included in the above-described screening method. Examples of the TRPA1 activator include allyl isothiocyanate, cinnamic aldehyde, and acrolein.

As used herein, “a substance that suppresses the activity of the channel” has the same definition as that for “a substance that inhibits the channel” to refer to a substance that suppresses the activation of the ion channel by being brought into contact with a test substance, including both a substance that inhibits channel activity, like a TRPA1 inhibitor, and a substance that increases the activity of a substance that directly inhibits the channel. By performing the above-described step in the presence of a TRPA1 inhibitor, a substance that promotes the inactivation of TRPA1 by a TRPA1 inhibitor can be screened for; a screening method for a substance that increases the inactivation of the aforementioned polypeptide by a TRPA1 inhibitor is also included in the above-described screening method. Examples of the TRPA1 inhibitor include Ruthenium Red.

Analysis of channel activity in the screening method of the present invention can be performed in a variety of modes. Examples of such modes include (a) utilization of the patch-clamp method, (b) utilization of radioisotope ion inflow, (c) utilization of an intracellular Ca²⁺ detection dye. The individual screening methods are hereinafter described.

When screening is performed by utilizing the patch-clamp method of (a), by, for example, analyzing (preferably measuring) the whole cell current in cells using the whole cell patch-clamp method (Hille, B., Ionic Channels of Excitable Membranes, 2nd Ed., 1992, Sinauer Associates Inc., MA), an analysis can be performed to determine whether or not the channel is activated.

More specifically, the screening tool cells of the present invention are subjected to membrane potential fixation by the whole cell patch-clamp method, and the whole cell current of the aforementioned cells is measured. In this case, as the extracellular fluid, a solution containing 149 mmol/L-NaCl, 5 mmol/L-KCl, 2 mmol/L-CaCl₂, 0.8 mmol/L-MgCl₂, and 10 mmol/L-HEPES-Na (pH 7.4) can be used, and as the intracellular fluid, a solution comprising 147 mmol/L-CsCl, 4.5 mmol/L-EGTA, and 9 mmol/L-HEPES-K (pH 7.2) and the like can be used. Subsequently, by measuring changes in current with the addition of a test substance to the extracellular fluid or intracellular fluid, a substance that activates the channel of the polypeptide or screening tool polypeptide of the present invention can be screened for. For example, if the changes in whole cell current upon stimulation by activation of the aforementioned channel intensify with the addition of a test substance, the aforementioned test substance can be judged to be a substance that activates the aforementioned channel. As a substance that activates the channel, it is preferable to select, for example, one that produces changes in cell current to the same extent as a TRPA1 activator as described in an Example.

When screening is performed by utilizing a radioisotope ion inflow of (b), channel activity can be analyzed (preferably measured) with various radioisotopes of Ca²⁺ ions as indexes [Sidney P. Colowick and Nathan O. Kaplan, Methods in ENZYMOLOGY, 88(1), 1982, Academic Press, 346-347]. This analytical procedure is based on the finding that the screening tool polypeptide of the present invention transmits Ca²⁺ ions.

In the screening tool cells of the present invention, by analyzing the amount of the radioactivity flowing into the aforementioned cells, or the radioactivity remaining outside the cells, using a test substance, whether or not the channel of the screening tool polypeptide of the present invention is activated can be determined.

Specifically, the amount of the radioactivity can be measured using, for example, ⁴⁵Ca²⁺, a radioisotope of Ca²⁺ ion. If a test substance activates the aforementioned channel in a state wherein ⁴⁵Ca²⁺ is in the reaction liquid, the radioisotope flows into the cells; therefore, the radioactivity in the extracellular fluid (that is, radioactivity remaining in the extracellular fluid), or the radioactivity of the radioisotope flowing into the cells, can be used as the index of channel activation (Toshio Kuroki, Huh, Nam-Ho, and Kazuhiro Chida edts., Jikken Igaku, extra issue, “Bunshi Seibutsugaku Kenkyu No Tameno Baiyou Saibou Jikkenhou”, 1995, Yodosha Co., Ltd.). As a substance that activates the channel, for example, one that allows Ca²⁺ to flow into cells to the same extent as a TRPA1 activator as described in an Example, specifically, one that has an EC₅₀of 100 μmol/L or less, is preferably selected.

When screening is performed by utilizing an intracellular Ca²⁺ detection dye of (c), for example, Fluo3-AM and the like can be used as an intracellular Ca²⁺ detection dye. The intracellular Ca²⁺ detection dye makes it possible to optically analyze (preferably measure) changes in intracellular Ca²⁺ concentration resulting from the opening of the ion channel of the screening tool polypeptide of the present invention (Yoshihisa Kudo edt., Jikken Igaku, extra issue, “Saibounai Karushiumu Jikken Purotokoru”, 1996, Yodosha Co., Ltd.). By using these dyes, the activity of the aforementioned channel can be measured. If the intracellular Ca²⁺ detection dye shows a change in the presence of a test substance compared to the finding obtained in the absence of the test substance, in the aforementioned channel expression cells, the test substance can be judged to be a substance that activates the aforementioned channel. This method is not particularly limited; for example, by allowing the screening tool cells of the present invention to incorporate an intracellular Ca²⁺ detection dye, and then optically measuring quantitative changes in the intracellular Ca²⁺ detection dye caused by the test substance in the aforementioned cells, whether or not the aforementioned channel is activated can be determined.

More specifically, if the amount of Ca²⁺ flowing into cells increases with the addition of a test substance compared to the amount obtained in the absence of the test substance, the aforementioned test substance can be judged to be a substance that activates the channel. This method is preferably performed under the conditions described in Examples 3, 4, 5, and 13; as a substance that activates the channel, for example, one that promotes quantitative changes in intracellular Ca²⁺ detection dye to the same extent as that caused by a TRPA1 activator as described in an Example, specifically, one having an EC₅₀of 100 μmol/L or less under the conditions of Example 4, is preferably selected. As a substance that inactivates the channel, for example, one that promotes quantitative changes in intracellular Ca²⁺ detection dye to the same extent as that caused by a TRPA1 inhibitor as described in an Example, specifically, one having an EC₅₀of 100 μmol/L or less under the conditions of Example 5, is preferably selected.

In the aforementioned screening method (a), (b), or (c), out of compounds that do not directly activate the aforementioned channel, one that exhibits a higher activity than that obtained without administration of the aforementioned test substance when a TRPA1 activator at a is concentration that does not 100% activate the aforementioned channel, for example, a TRPA1 activator at 1 μmol/L, is administered after administration of the aforementioned test substance, can be judged to promote the activity of the aforementioned channel. As stated above, by performing the above-described screening in the presence of a TRPA1 activator, a compound that promotes the activation of the screening tool polypeptide of the present invention by the TRPA1 activator can be screened for. As a substance that promotes the activation, one that significantly promotes the activity of the TRPA1 activator, specifically, one having an EC₅₀of 100 μmol/L or less, is preferably selected.

For inhibitor screening methods as well, in the aforementioned screening method (a), (b), or (c), out of compounds that do not directly inactivate the aforementioned channel, one that exhibits a higher inhibitory activity than that obtained without administration of the aforementioned test substance when a TRPA1 inhibitor at a concentration that does not completely inactivate the aforementioned channel, for example, a TRPA1 inhibitor at 100 nmol/L, is administered after administration of the aforementioned test substance, can be judged to inactivate the activity of the channel. As stated above, by performing the above-described screening in the presence of a TRPA1 inhibitor, a compound that increases the inactivation of the screening tool polypeptide of the present invention by the TRPA1 inhibitor can be screened for. As a substance that promotes the inactivation, one that significantly promotes the inhibitory activity of the TRPA1 inhibitor, specifically, one having an EC₅₀of 100 μmol/L or less, is preferably selected.

When a screening polypeptide that exhibits cesium, sodium, or magnesium ion transmitting ion channel activity is used, a radioisotope of cesium, sodium, or magnesium can be used as is the indicator in place of Ca²⁺ as in (b). Specifically, the screening can be performed with reference to the method described in Sidney P. Colowick and Nathan O. Kaplan, Methods in ENZYMOLOGY, 88(1), 1982, Academic Press, 346-347. This analytical procedure is based on the finding that the screening polypeptide of the present invention transmits cesium ions, sodium ions, and magnesium ions.

The screening method I of the present invention can be useful in developing a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, including digestive organ diseases, as described above. For example, a substance capable of promoting the expression or channel activity of TRPA1 can be useful as a prophylactic or therapeutic drug for constipation type irritable bowel syndrome, functional dyspepsia or constipation among digestive organ diseases, and also as a prophylactic/therapeutic drug for bulimia, insomnia, depression, anxiety disorders, migraine, and platelet aggregation dysfunction among non-digestive organ diseases. On the other hand, a substance capable of suppressing the expression or channel activity of TRPA1 can be useful as, for example, a prophylactic or therapeutic drug for diarrhea type irritable bowel syndrome, diarrhea or vomiting among digestive organ diseases, and also as a prophylactic/therapeutic drug for anorexia nervosa, pain, schizophrenia, carcinoid tumor, thrombosis, and pulmonary thromboembolism among non-digestive organ diseases.

As the screening method I of the present invention, a screening method comprising selecting a substance that binds to TRPA1, comprising the following steps (a) to (c), can also be mentioned:

(a) a step for bringing a test substance into contact with the polypeptide type screening tool of the present invention;
(b) a step for analyzing the binding of the aforementioned test substance to the aforementioned screening tool; and
(c) a step for selecting a substance that binds to the aforementioned screening tool.

The screening method I of the present invention, in addition to the aforementioned steps (a) to (c), may further comprise as the step (d) a step for determining whether or not the selected substance is effective as a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, or a step for determining whether or not the selected substance is effective as a prophylactic and/or therapeutic drug for digestive tract diseases. This confirmatory step can be performed by using a method obvious to those skilled in the art, or a method improved therefrom. Examples include a test to measure gut movement using animals, measurement of the amount of defecation, measurement of fecal nature, measurement of contraction using an isolated gut, measurement of the amount of gut water secretion and the like as described in Examples 19 to 22 below.

2.2. Screening Method Comprising Selecting a Substance that Does Not Regulate the Expression or Channel Activity of TRPA1

The present invention provides a screening method comprising evaluating a test substance to determine whether or not the test substance is capable of regulating the expression or channel activity of TRPA1, and selecting a substance that does not regulate the expression or channel activity of TRPA1 (screening method II).

The test substance subjected to the screening method II is not particularly limited, as far as it exhibits a specified pharmacological effect (e.g., drugs, bioactive substances); for example, the above-described test substances can be used. An analysis of the expression or channel activity of TRPA1 in the screening method II of the present invention can be performed in the same manner as the screening method I. The screening method II of the present invention can be useful in developing a pharmaceutical that exhibits a specified pharmacological effect, and that is not desired to act as a result of the capability of regulating 5-HT release (e.g., adverse reactions in digestive organs) (e.g., pharmaceuticals with a decreased incidence of adverse reactions).

3. Pharmaceutical

The present invention provides a pharmaceutical composition containing a substance capable of regulating the expression or channel activity of the screening tool polypeptide of the present invention, for example, a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, including digestive organ diseases.

The present invention also provides a prophylactic and/or therapeutic method for diseases associated with 5-HT production/secretion abnormalities, including digestive organ disease, comprising administering a substance capable of regulating the expression or channel activity of a screening tool polypeptide, and a use of a substance capable of regulating the expression or channel activity of the screening tool polypeptide of the present invention for producing a pharmaceutical composition.

The present invention further provides a method of producing a pharmaceutical composition, comprising evaluating a test substance to determine whether or not the test substance is capable of regulating the expression or channel activity of TRPA1, and preparing the evaluated substance as a pharmaceutical preparation, and a pharmaceutical composition is obtained by the method of production.

In an embodiment, the method of production of the present invention can be a method of producing a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, including digestive organ diseases, comprising screening for a substance capable of regulating the expression or channel activity of TRPA1, and preparing the substance obtained by the screening as a pharmaceutical preparation (method of production I).

In more detail, the method of production I of the present invention can comprise the following steps (a) to (d):

(a) a step for bringing a test substance into contact with the screening tool of the present invention;
(b) a step for analyzing the expression or channel activity of TRPA1; and
(c) a step for selecting a substance capable of regulating the expression or channel activity of TRPA1;
(d) a step for preparing the substance obtained in the step (c) as a pharmaceutical preparation.

In another embodiment, the method of production of the present invention can be a method of producing a pharmaceutical composition, comprising screening for a substance that exhibits a specified pharmacological effect, and that does not regulate the expression or channel activity of TRPA1, and preparing the substance obtained by the screening as a pharmaceutical preparation (method of production II).

In more detail, the method of production II of the present invention can comprise the following steps (a) to (d):

(a) a step for bringing a test substance that exhibits a specified pharmacological effect into contact with the screening tool of the present invention;
(b) a step for analyzing the expression or channel activity of TRPA1 and
(c) a step for selecting a substance that does not regulate the expression or channel activity of TRPA1, and that exhibits a specified pharmacological effect;
(d) a step for preparing the substance obtained in the step (c) as a pharmaceutical preparation.

The steps (a) to (c) in the method of production I and method of production II of the present invention can be performed in the same manner as the screening method of the present invention.

A substance selected through the above-described steps (a) to (c) in the method of production of the present invention [for example, DNAs, proteins (including antibodies or antibody fragments), peptides, or other compounds] can be prepared as a pharmaceutical preparation using a pharmacologically acceptable carrier, excipient, and/or other additives in common use in the art chosen according to the kind thereof, as a pharmaceutical composition.

Examples of modes of administration include oral administration of tablets, pills, capsules, granules, fine granules, powders, or solutions for oral administration and the like, or parenteral administration of injections such as intravenous injection or intramuscular injection, suppositories, transdermal preparations, or per-mucosal preparations and the like. In particular, for peptides that are digested in the stomach, parenteral administration by intravenous injection and the like is preferred.

In the solid composition for oral administration, one or more active substances and at least one inactive diluent, for example, lactose, mannitol, glucose, microcrystalline cellulose, hydroxypropylcellulose, starch, polyvinylpyrrolidone, or magnesium metasilicate aluminate and the like can be blended. The aforementioned composition can contain, in accordance with a conventional method, an additive other than an inactive diluent, for example, a lubricant, a disintegrant, a stabilizer, or a solvent or solubilizer and the like. Tablets or pills can be coated with a sugar coating or a film such as of a substance that dissolves in the stomach or intestine as required.

The liquid composition for oral administration can contain, for example, an emulsion, a solution, a suspension, a syrup, or an elixir, and can contain an inactive diluent in general use, for example, purified water or ethanol. The aforementioned composition can contain an additive other than an inactive diluent, for example, a wetting agent, a suspending agent, a sweetening agent, a flavoring agents, or an antiseptic.

Injections for parenteral administration can contain a sterile, aqueous or non-aqueous solution, suspension, or emulsion. The aqueous solution or suspension can contain, for example, distilled water for injection or physiological saline and the like as a diluent. As examples of the diluent for the non-aqueous solution or suspension, propylene glycol, polyethylene glycol, vegetable oils (for example, olive oil), alcohols (for example, ethanol), or polysorbate 80 and the like can be contained. The aforementioned composition can further contain a wetting agent, an emulsifier, a dispersing agent, a stabilizer, a solvent or a solubilizer, or an antiseptic and the like. The aforementioned composition can be sterilized by, for example, filtration through a bacterial retention filter, formulation of an antibacterial agent, or irradiation. It is also possible to produce a sterile solid composition and dissolve it in sterile water or another medium for sterile injection before use.

The dose can be determined as appropriate in consideration of the potency of the activity of an active ingredient, symptoms, subject age or sex and the like.

For example, in the case of oral administration, the dose is normally about 0.1 to 100 mg, preferably 0.1 to 50 mg, per day for an adult (assuming a body weight of 60 kg). In the case of parenteral administration, in the form of an injection, the dose is 0.01 to 50 mg, preferably 0.01 to 10 mg, per day.

Examples

The present invention is hereinafter described in further detail by means of the following examples, which, however, do not limit the scope of the present invention.

Example 1 Isolation of Human-Derived TRPA1 and Construction of Expression Vector

After 10 ng of human brain mRNA (Clontech) was treated with DNase, reverse transcription was performed using a kit for reverse transcription-polymerase chain reaction (RT-PCR) (SUPERSCRIPT First-Strand Synthesis System for RT-PCR; Invitrogen) to synthesize a first strand cDNA. With this first strand cDNA as the template, using Taq DNA polymerase (LA Taq DNA polymerase; Takara Shuzo), PCR was performed by the Hot Start method. The aforementioned PCR was performed using oligonucleotides consisting of the base sequences shown by SEQ ID NO:7 as a sense primer, and SEQ ID NO:8 as an antisense primer; first, thermal denaturation was performed at 98° C. (1 minute), after which a cycle consisting of heat treatment at 98° C. (15 seconds)/56° C. (30 seconds)/72° C. (5 minutes) was repeated 35 times. As a result, an about 3.3-kbp DNA fragment was amplified.

This DNA fragment was cloned into the pCR-TOPO vector using a cloning kit (TOPO XL PCR Cloning Kit; Invitrogen). The plasmid DNA obtained was digested with the restriction endonucleases KpnI and HindIII, after which it was cloned using the plasmid pcDNA3.1(+) (Invitrogen). The aforementioned plasmid pcDNA3.1(+) has a cytomegalovirus-derived promoter sequence, and can be used to express a protein in animal cells.

When the base sequence of the clone obtained was analyzed by the dideoxy terminator method using a DNA sequencer (ABI3700 DNA Sequencer; Applied Biosystems), the base sequence shown by SEQ ID NO:1 was obtained. When these sequences were translated into amino acid sequences, the amino acid sequence shown by SEQ ID NO:2 was obtained.

Example 2 Expression of Protein in Animal Cells

To detect the TRPA1 channel activity of a polypeptide consisting of the amino acid sequence shown by SEQ ID NO:2, the expression vector obtained in Example 1 above was transfected to animal cells, whereby the aforementioned protein was expressed. Fetal human kidney-derived HEK293 cells and CHO-K1 cells were transformed using the expression vector obtained in Example 1 and a transformation reagent (LIPOFECTAMINE or LIPOFECTAMINE2000; Invitrogen) to induce the expression of the polypeptide consisting of the amino acid sequence shown by SEQ ID NO:2.

The aforementioned operation was performed per the protocol attached to the aforementioned transformation reagent, and a commonly known method (Toshio Kuroki, Huh, Nam-Ho, and Kazuhiro Chida edts., Jikken Igaku, extra issue, “Bunshi Seibutsugaku Kenkyu No Tameno Baiyou Saibou Jikkenhou”, 1995, Yodosha Co., Ltd.).

Example 3 Measurement of Intracellular Calcium Concentrations by FLIPR

Various test samples were added to CHO-K1 cells forced to is transiently express TRPA1 by the transfection operation of Example 2 above, and the resulting changes in intracellular calcium concentration were measured using FLIPR (Molecular Device).

To measure the changes in intracellular calcium concentration by FLIPR, the following pre-treatment was performed. First, an assay buffer for adding the fluorescent dye Fluo3-AM (DOJIN) to the cells, or for washing the cells just before performing the FLIPR assay, was prepared. To a solution prepared by adding 20 ml of 1M HEPES (pH 7.4) (Invitrogen) to 1000 ml of HBSS (Invitrogen) (hereinafter, HBSS/HEPES solution), 10 ml of a solution prepared by dissolving 710 mg of probenecid (Sigma) in 5 ml of 1N NaOH and then adding 5 ml of the HBSS/HEPES solution, was added and mixed, and this solution was used as the assay buffer. Next, 50 μg of Fluo3-AM was dissolved in 22 μl of DMSO (DOJIN), and an equal volume of 20% pluronic acid (Molecular Probes) was added and mixed, after which this mixture was added to 10.6 ml of the assay buffer supplemented with 105 μl of fetal bovine serum, whereby a fluorescent dye solution was prepared. The medium for the transfection-treated CHO-K1 cells was removed, and the fluorescent dye solution was immediately dispensed at 100 μl per well, after which the cells were cultured in a CO₂incubator for 1 hour to allow the cells to incorporate the fluorescent dye. After the cultivation, the cells were washed with the above-described assay buffer, and then set to the FLIPR. A test sample for addition to the TRPA1-expressing CHO-K1 cells was prepared using the assay buffer, and simultaneously set to the FLIPR. After this pretreatment was performed, changes in intracellular calcium concentration after addition of the various test samples were measured with the FLIPR.

As a result, it was found that when allyl isothiocyanate (Wako Pure Chemical Industries), cinnamic aldehyde (Wako Pure Chemical Industries), acrolein (Sigma) and the like were added, CHO-K1 cells expressing human TRPA1 responded specifically (elevation of intracellular calcium concentration). On the other hand, in an investigation using CHO-K1 cells not expressing the polypeptide shown by SEQ ID NO:2 (negative control cells), none of these compounds produced an elevation of fluorescence intensity. Hence, it was confirmed that allyl isothiocyanate, cinnamic aldehyde, and acrolein are activators of human TRPA1 (FIG. 1).

Example 4 Screening for TRPA1 Activators

Compounds that activate a polypeptide consisting of the amino acid sequence shown by SEQ ID NO:2 (activators) were screened for. As an index of activation, calcium inflow in the cells was detected using a calcium-sensitive fluorescent reagent; specifically, the method described in Example 3 was used. As the screening criterion, compounds having an EC₅₀of 100 μmol/L or less were selected.

As a result of investigations of various compounds, an elevation of fluorescence intensity was detected with allyl isothiocyanate, cinnamic aldehyde, and acrolein. The activation of the polypeptide consisting of the amino acid sequence shown by SEQ ID NO:2 by each compound was 17.1 μmol/L, 22.5 μmol/L, and 7.0 μmol/L, respectively, in terms of EC₅₀.

From these results, it was found that allyl isothiocyanate, cinnamic aldehyde, and acrolein have the action of activating the polypeptide consisting of the amino acid sequence shown by SEQ ID NO:2 to allow calcium to flow into cells.

Example 5 Screening for TRPA1 Inhibitors

Compounds that inhibit a polypeptide consisting of the amino acid sequence shown by SEQ ID NO:2 (inhibitor) were screened for. Inhibitory activity was measured by performing detection of calcium inflow in the cells using a calcium-sensitive fluorescent reagent; specifically, the method described in Example 3 was used with a modification. As the screening criterion, compounds having an IC₅₀of 100 μmol/L or less were selected. For inhibitor measurements, various compounds at 30 μM (final concentration at the time of reaction was 10 μM) were dispensed to a plate, and the plate was simultaneously set to FLIPR. After this pretreatment was performed, changes in intracellular calcium concentration after addition of cinnamic aldehyde were measured by with the FLIPR, and their inhibitory actions were investigated. Ruthenium Red was found to be a compound that inhibits the elevation of fluorescence intensity. The inhibitory activity of Ruthenium Red on the polypeptide consisting of the amino acid sequence shown by SEQ ID NO:2 was 2.2 μmol/L in terms of IC₅₀.

Example 6 Expression Analysis in Human Tissues

The expression of the TRPA1 gene in human tissue was analyzed by real time PCR using a sequence detector (PRISM7900; Applied Biosystems). By performing real time PCR, the desired gene contained in mRNA can be quantitatively measured.

From 1 μg of polyA⁺RNA (CLONTECH Laboratories) derived from various human tissues, a reverse transcription reaction was carried out using random primers. A cDNA obtained by carrying out the reaction using the reverse transcriptase SuperScript II (GIBCO BRL) per the attached protocol was used in the experiment. With this first strand cDNA as the template, using a fluorescent reagent (SYBR Green PCR Core Reagents Kit; Applied Biosystems), PCR was performed. The aforementioned PCR was performed using an oligonucleotide consisting of the base sequence shown by SEQ ID NO:9 as a sense primer, and an oligonucleotide consisting of the base sequence shown by SEQ ID NO:10 as an antisense primer; first, thermal denaturation was performed at 95° C. (10 minutes), after which a cycle consisting of heat treatment at 95° C. (15 seconds)/59° C. (1 minute) was repeated 45 times. Each primer is a sequence specific for a gene consisting of the base sequence shown by SEQ ID NO:1.

The distributions of mRNA expression in various human tissues are shown in FIG. 2. High expression was detected in the stomach, small intestine, large intestine, urinary bladder and the like. From this finding, it was demonstrated that the mRNA consisting of the base sequence shown by SEQ ID NO:1 is expressed in digestive tissues such as the stomach, small intestine, and large intestine, and that the polypeptide consisting of the amino acid sequence shown by SEQ ID NO:2 functions in digestive tissues such as the stomach, small intestine, and large intestine.

Example 7 Expression Analysis in Mouse Tissues

RNA was prepared from mouse tissues as described below. A C57BL6 mouse (male, 8-week-old) was decapitated and exsanguinated, after which it was dissected with scissors, and the brain, stomach, small intestine, and large intestine were extirpated. These tissues were washed with ice-cooled physiological saline, after which they were homogenized by the addition of Isogen (Nippon Gene Co., Ltd.), and total RNA was prepared per the manual. For 1 μg of the extracted RNA, a first strand cDNA was synthesized using random primers per the manual of SuperScript II (Invitrogen), after which it was dissolved in 200 μl of TE.

Example 8 Expression Analysis in Mouse Tissues (Real Time PCR)

The expression of the TRPA1 gene in mouse tissues was analyzed by real time PCR using a sequence detector (PRISM7900; Applied Biosystems). With the mouse tissue first strand cDNA obtained in Example 7 above as the template, using a fluorescent reagent (SYBR Green PCR Core Reagents Kit; Applied Biosystems), PCR was performed. The aforementioned PCR was performed using an oligonucleotide consisting of the base sequence shown by SEQ ID NO:11 as a sense primer, and an oligonucleotide consisting of the base sequence shown by SEQ ID NO:12 as an antisense primer; first, thermal denaturation was performed at 95° C. (10 minutes), after which a cycle consisting of heat treatment at 95° C. (15 seconds)/59° C. (1 minute) was repeated 45 times. Each primer is a sequence specific for a gene consisting of the base sequence shown by SEQ ID NO:3. When these base sequences are translated, the amino acid sequence shown by SEQ ID NO:4 is obtained.

As a result, relative to the expression level of a reference standard of the mouse β actin gene as 100%, in the mouse stomach, jejunum, and large intestine, 0.037%, 0.084%, and 0.094%, respectively, of TRPA1 mRNA expressions were observed, whereas the expression level in the whole brain was 0.014%. From this finding, it was demonstrated that the TRPA1 mRNA consisting of the base sequence shown by SEQ ID NO:3 is expressed in the digestive tissues, and that a polypeptide consisting of the amino acid sequence shown by SEQ ID NO:4 is functioning.

Example 9 Expression Analysis in Rat Tissues (1) Rat Tissues

RNA was prepared from rat tissues as described below. A Wistar rat (male, 8-week-old) was decapitated and exsanguinated, after which it was dissected with scissors, and the brain, small intestine, and large intestine were extirpated. These tissues were washed with ice-cooled physiological saline, and the small intestine and large intestine were separated into the mucosal layer and the smooth muscle layer using glass slides. These tissue samples were homogenized by the addition of Isogen (Nippon Gene Co., Ltd.), and total RNA was prepared per the manual. For 1 μg of the extracted RNA, a first strand cDNA was synthesized using random primers per the manual of SuperScript II (Invitrogen), after which it was dissolved in 200 μl of TE.

(2) Cultured Cells and Medium

RIN14B cells (rat pancrease-derived endocrine cell line) were purchased from ATCC. The RIN14B cells were cultured using an RPMI1640 medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) unless otherwise stated. The RIN14B cells were cultured using an RPMI1640 medium (Invitrogen) containing 10% FCS until they became pre-confluent, and they were used for experiments such as gene expression analysis.

(3) Isolation of Rat Small Intestine EC Cells

A Wistar rat (male, 8-week-old) was decapitated and exsanguinated, after which it was dissected and laparotomized with scissors, and the small intestine was extirpated. The inside of the lumen of the extirpated small intestine was washed with physiological saline, and about 20 mL of Buffer A (70 mM NaCl, 5 mM KCl, 20 mM NaHCO₃, 0.5 mM NaH2PO4, 50 mM HEPES (pH 7.2), 11 mM glucose, 3 mM EDTA, 0.5% BSA, 0.05 mM dithiothreitol, 1 mg/mL N-acetyl-L-cysteine) was injected, after which both ends were closed, and the small intestine was allowed to stand in 37° C. incubated HBSS for 10 minutes. Thereafter, the Buffer A in the lumen of the small intestine was discarded, about 20 mL of Buffer A was injected again, and the small intestine was allowed to stand in 37° C. incubated HBSS for 10 minutes. Again, the Buffer A in the lumen of the small intestine was discarded, fresh Buffer A was injected, and the small intestine was allowed to stand in 37° C. incubated HBSS for 20 minutes, after which the lumen content was recovered. This operation was repeated three times in total, all the lumen contents were combined together, and this was used as the small intestine mucosal epithelial cell sample.

Next, an EC cell fraction was prepared using counterflow centrifugal elutriation (CCE). A CCE apparatus (BECKMAN, JE-5.0) was operated at a fixed rotor speed of 2000 rpm, with a PBS containing 1% fetal bovine serum, 1% glucose, 1 mM dithiothreitol, and 1 mM EDTA used as the buffer for CCE. A small intestine mucosal epithelial cell sample was injected to the CCE apparatus, and the cells flowing out at 21 mL/min were recovered, after which the sample was further purified by density gradient centrifugation using a Percoll solution (d=1.132 g/mL, Pharmacia). A 9-fold volume of the Percoll solution was added to a 10-fold concentration of HBSS, and this was used as the 100% Percoll solution. The 100% Percoll was diluted with a 1-fold concentration of HBSS to yield a 60% Percoll solution, a 30% Percoll solution, and a 20% Percoll solution, which were overlain in a centrifugal tube. Furthermore, the CCE-purified sample was overlain thereon, and centrifuged at 1100 rpm for 10 minutes. The cells gathering in the interface between the 60% Percoll solution and the 30% Percoll solution were recovered and washed with PBS, and this was used as the EC cell fraction. This EC cell fraction was assayed to determine the expression levels of the marker genes for TPH1, chromogranin A, synaptophysin, and VMAT1 by real time PCR method; samples confirmed to exhibit marker gene expression levels not less than 20 times higher than the level for a small intestine mucosal epithelial cell sample were used in the experiments that followed (Table 1).

The aforementioned operation was performed per the protocol attached to the elutriator system, and a commonly known method (Shunsuke Migita edt., “Men-eki Jikken Sousahou 2”, 1995, Nankodo).

(4) Extraction of RNA and Synthesis of cDNA

For the RIN14B cells and the rat EC cell fraction, the cells were isolated and counted, after which total RNA was extracted and purified per the manual of the RNeasy mini KIT (QIAGEN). For 1 μg of the extracted RNA, a first strand cDNA was synthesized using random primers per the manual of SuperScript II (Invitrogen), after which it was dissolved in 200 μl of TE.

Example 10 Expression Analysis in Rat Tissues (Real Time PCR)

The expression of the TRPA1 gene in rat tissues, a rat EC cell fraction and RIN14B cells was analyzed by real time PCR using a sequence detector (PRISM7900; Applied Biosystems). With the first strand cDNA derived from rat tissue, rat EC cells or RIN14B cells, obtained in the aforementioned Example, as the template, using a fluorescent reagent (SYBR Green PCR Core Reagents Kit; Applied Biosystems), PCR was performed. The aforementioned PCR was performed using an oligonucleotide consisting of the base sequence shown by SEQ ID NO:13 as a sense primer, and an oligonucleotide consisting of the base sequence shown by SEQ ID NO:14 as an antisense primer; first, thermal denaturation was performed at 95° C. (10 minutes), after which a cycle consisting of heat treatment at 95° C. (15 seconds)/59° C. (1 minute) was repeated 45 times. Each primer is a sequence specific for a gene consisting of the base sequence shown by SEQ ID NO:5. When these sequences are translated into amino acid sequences, the amino acid sequence shown by SEQ ID NO:6 is obtained.

As a result, relative to the expression level of a reference standard of the rat G3PDH gene as 100%, in the rat large intestine mucosa and small intestine mucosa, 0.79% and 0.85%, respectively, of TRPA1 mRNA expressions were observed, whereas the expression level in the brain was 0.11%. From this finding, it was demonstrated that TRPA1 is highly expressed in the rat gut and functions. In small intestine EC cells, a high expression level of TRPA1 mRNA was detected as with other EC cell markers, demonstrating that TRPA1 is expressed in the EC cells (Table 1). Furthermore, the RIN14B cells showed expression of the EC cell marker gene, and the TRPA1 gene, to equivalent or higher extent than the EC cells, demonstrating that RIN14B cells have properties very similar to those of EC cells (Table 2).

TABLE 1 Expression levels of TRPA1 mRNA and EC cell marker genes in rat small intestine EC cell fraction Ratio (% of small intestine mucosal tissue) Small intestine mucosal tissue EC cell fraction TPH1 100 7310.5 Chromogranin A 100 7837.2 VMAT 1 100 3777.6 Synaptophysin 100 2288.4 TRPA1 100 1626.7

The values are relative to the expression level of each gene in rat small intestine mucosal tissue as 100%.

TABLE 2 Expression levels of TRPA1 mRNA and EC cell marker genes in digestive endocrine cell-derived RIN14B cell line (ratio) TPH1 chromograinA VMAT1 synaptophysin TRPA1 Small cell 100.0 100.0 100.0 100.0 100.0 EC cell fraction RIN14B 917.4 72646.8 3442.6 98.8 159.7

The values are relative to the expression level of each gene in rat EC cell fraction as 100%.

Example 11 Expression Analysis in Human Tissue (In Situ Hybridization/Immunohistochemical Staining)

To confirm the expression of TRPA1 in human EC cells, in situ hybridization staining was performed using the human duodenum.

Paraffin-embedded human duodenum tissue (CYTOMYX) was sectioned to 6 μm thickness, and this was used as the sample for in situ hybridization staining.

With the plasmid pcDNA-human TRPA1 obtained in Example 1 as the template, by the in vitro transcription method, a digoxigenin-labeled RNA antisense probe was prepared. Digoxigenin labeling was performed using a commercially available reagent (DIG RNA Labeling Mix; Roche) per the attached protocol. For negative control, using the same method, a digoxigenin-labeled RNA sense probe was also prepared. The probe sequence used was the same region as the 2870th to 3360th base sequence of the human TRPA1 gene sequence shown by SEQ ID NO:1.

Using the sample and probe obtained above, in situ hybridization staining was performed. The antibody used was an alkaline phosphatase-labeled anti-digoxigenin antibody (Roche), and the color development substrate used was NBT/BCI (mixed liquid of 5-bromo-4-chloro-3-indoylphosphoric acid and nitro blue tetrazolium salt); after color development, nuclear staining was performed with kernechtrot.

As a result, in the investigation using the antisense probe, intense color development was observed specifically in some cells in the epithelium of the human duodenum. In the investigation using the sense probe, no staining was observed. From these results, it was demonstrated that the human TRPA1 gene, which consists of the base sequence shown by SEQ ID NO:1, is expressed in the cells in the gut lacuna also in humans (FIG. 3).

Example 12 Expression Analysis in Human Tissue (In Situ Hybridization/Immunohistochemical Staining)

To determine whether or not the TRPA1 expression site observed in Example 11 was EC cells, in situ hybridization is staining was performed using the human duodenum, after which immune staining was performed with an anti-serotonin antibody.

After TRPA1 was stained by in situ hybridization of the human duodenum by the same method as Example 11, a reaction was carried out using an anti-serotonin antibody (Sigma) as the primary antibody. Furthermore, a reaction was carried out using a biotinized anti-rabbit IgG antibody as the secondary antibody, after which a color developing reaction was carried out using DAB as the color development substrate. As a result, the epithelial cells of the human duodenum, which showed the expression of TRPA1, also showed color development by the serotonin antibody. From these results, it was demonstrated that the TRPA1 gene, consisting of the base sequence shown by SEQ ID NO:1, is expressed in the serotonin-expressing epithelial cells of the human duodenum, that is, EC cells (FIG. 4).

Example 13 Detection of Channel Activity in RIN14B Cells Using Calcium-Sensitive Fluorescent Reagent

RIN14B cells (5×10⁴cells), wherein the expression of TRPA1 was confirmed in Example 10, were incubated in the presence of a calcium-sensitive fluorescent reagent (Fluo3-AM; DOJINDO) at 37° C. for 1 hour to thereby incorporate the calcium-sensitive fluorescent reagent in the cells, after which the cells were washed with physiological saline to remove the calcium-sensitive fluorescent reagent that had not been incorporated in the cells. To the cells obtained, physiological saline supplemented with allyl isothiocyanate, cinnamic aldehyde, or acrolein was added; the fluorescence emitted by the cells was measured over time. The above-described measurements were performed using an automated fluorescence detection apparatus (FLIPR; Molecular Device). Using physiological saline not supplemented with allyl isothiocyanate, cinnamic aldehyde, or acrolein, the same operation was performed. Ruthenium Red was added concurrently and a measurement was performed to determine whether or not calcium inflow in the cells was be inhibited.

As a result, in RIN14B cells having allyl isothiocyanate, cinnamic aldehyde, or acrolein added thereto, an elevation of fluorescence intensity was detected from soon after the addition. On the other hand, in the investigation using physiological saline not supplemented with allyl isothiocyanate, cinnamic aldehyde, or acrolein, no fluorescence was detected in any case. This shows that TRPA1 was activated by allyl isothiocyanate, cinnamic aldehyde, and acrolein to allow calcium to flow into the cells. Furthermore, changes in intracellular Ca²⁺ concentration with the addition of various concentrations of allyl isothiocyanate, cinnamic aldehyde, and acrolein to RIN14B cells were examined; it was demonstrated that they concentration-dependently allow calcium to flow into the cells.

When Ruthenium Red was added, the elevation of fluorescence intensity was inhibited by allyl isothiocyanate, cinnamic aldehyde, and acrolein (30 μM Ruthenium Red 90.9% inhibited the activation by allyl isothiocyanate).

Example 14 Measurements of Serotonin Secretion from RIN14B

To determine whether or not TRPA1 is involved in serotonin release, the promotion of serotonin secretion from RIN14B by TRPA1 activators was measured.

After RIN14B cells in culture in a Petri dish were detached using a PBS containing 1 mM EDTA, they were sown to a 96-well plate and cultured for 2 days. The medium used was RPMI1640 (Invitrogen Japan K.K.) supplemented with 10% fetal bovine serum (ICN), 100 U/ml penicillin, and 100 μg/ml streptomycin. After the cells were once washed with Hanks' Balanced Salt Solutions (HBSS, Invitrogen) supplemented with 0.1% BSA and 10 μM fluoxetine (TOCRIS), each TRPA1 activator, previously diluted/prepared with the above-described HBSS, was added, and the RIN14B cells were cultured at 37° C. in the presence of 5% CO₂for 20 minutes. After the cultivation, the cell supernatant was recovered, and stored under freezing. The serotonin content in the supernatant was measured using a commercially available serotonin immunoassay kit (Beckman).

As a result, as shown in FIG. 5, serotonin secretion was promoted by allyl isothiocyanate, cinnamic aldehyde, and acrolein, all of which exhibited remarkable activities in an intracellular calcium ion inflow assay using RIN14B cells. On the other hand, when the cells were treated with Ruthenium Red concurrently with acrolein (30 μM), Ruthenium Red concentration-dependently suppressed acrolein-induced serotonin secretion (73.0% inhibited by 30 μM Ruthenium Red); when the cells were treated with Ruthenium Red (30 μM) concurrently with cinnamic aldehyde (30 μM), Ruthenium Red completely suppressed cinnamic aldehyde-induced serotonin secretion. From these results, it was demonstrated that TRPA1 is involved in the action of promoting serotonin secretion from RIN14B cells.

Example 15 Suppression of Expression of the Rat TRPA1 Gene by Introduction of an siRNA Specific for Rat TRPA1 Sequence

RIN14B cells were sown to a 60 mm Petri dish at 6×10⁵cells and cultured for 1 day. After various sequences of siRNA for rat TRPA1 (10 nM), designed using the siRNA design system siDirect (RNAi), were introduced using a transformation reagent (LIPOFECTAMINE2000; Invitrogen Japan K.K.), the RIN14B cells were further cultured for 2 days, and the expression level of the rat TRPA1 gene was measured. Detection of the expression level of the rat TRPA1 gene was attempted by the method of Example 10. As a result, by adding #971, a rat TRPA1-specific siRNA (sense strand was SEQ ID NO:15, antisense strand was SEQ ID NO:16), to the RIN14B cells, a reduction in the expression level of the rat TRPA1 was observed. From this finding, it was found that #971 specifically suppressed the expression of the rat TRPA1 gene.

Example 16 Suppressive Effect on the Intracellular Calcium Inflow Activity of Allyl isothiocyanate in siRNA-Introduced RIN14B

In Example 15, it was confirmed that #971, a TRPA1-specific siRNA, remarkably suppressed the expression of rat TRPA1. An investigation was performed on intracellular calcium inflow activity in RIN14B cells having #971 introduced thereto by the method of Example 15. As a result of an examination of the intracellular calcium inflow activity of allyl isothiocyanate by the method of Example 13, in RIN14B having #971 introduced thereto, the intracellular calcium inflow activity of allyl isothiocyanate (300 μM) was suppressed by 67.3%. On the other hand, it was shown that in RIN14B having the negative control siRNA, a random sequence siRNA, introduced thereto, the intracellular calcium inflow activity of the above-described activator was retained. From this result as well, it was confirmed that TRPA1 is also involved in the intracellular calcium inflow activity of allyl isothiocyanate.

Example 17 Suppressive Effect on the Serotonin Secretion Promoting Activity of Cinnamic Aldehyde in siRNA-Introduced RIN14B

In Example 16, it was confirmed that #971, a TRPA1-specific siRNA, remarkably suppressed the expression of rat TRPA1, and also suppressed intracellular calcium inflow. Hence, in RIN14B cells having #971 introduced thereto by the method of Example 15, serotonin secretion increasing activity was investigated. As a result of an examination of the serotonin secretion increasing activity of cinnamic aldehyde by the method of Example 14, as shown in FIG. 6, in RIN14B having #971 introduced thereto, the serotonin secretion increasing activity of cinnamic aldehyde was suppressed. On the other hand, in RIN14B having the negative control siRNA, a random sequence siRNA, introduced thereto, it was shown that the serotonin secretion promoting activity of the above-described activator was retained. From this result, it was proven that TRPA1 is involved in the promotion of serotonin secretion.

Example 18 Measurements of Serotonin Secretion from Rat EC Cells

Serotonin secretion activity in EC cells prepared by the method described in Example 9-(3) was measured by a method modified from the method of Example 14 above. A prepared rat EC cell fraction was once washed with a Hanks' Balanced Salt Solution (HBSS, Invitrogen) supplemented with 0.1% BSA, after which a TRPA1 activator, previously diluted/prepared with the above-described HBSS, was added, and the cells were cultured at 37° C. in the presence of 5% CO₂for 45 minutes. After the cultivation, the cell supernatant was recovered and stored under freezing.

The serotonin content in the supernatant was measured using a commercially available serotonin immunoassay kit (Beckman). As a result, as shown in FIG. 7, in rat EC cells, like in RIN14B, significant serotonin secretion promoting activity was observed with allyl isothiocyanate and cinnamic aldehyde. From the results above, it was proven that TRPA1 is responsible for the action of promoting serotonin secretion not only from RIN14B cells, but also from EC cells.

Example 19 Measurements of Isolated Guinea Pig Gut Constriction Activity

Guinea pigs (Hartley strain, male, weighing 300-400 g), under ether anesthesia, were exsanguinated to death by cutting the carotid artery. The ileum was extirpated, about 15-cm portions at both ends were removed, a section 1.5 cm long was cut out from the remaining portion, and incisions were made longitudinally parallelly in the gut to prepare a tabular specimen. This specimen was sandwiched with serrefine at both ends, and suspended with a thread in a Magnus chamber containing 10 ml of a 37° C. Krebs solution (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 11 mM D-glucose, 20 mM NaHCO₃) aerated with 95% O₂-5% CO₂mixed gas. A 1-g load was applied to the specimen, buffers were exchanged at 15-minute intervals, and the specimen was allowed to stand for about 60 minutes to stabilize its tension. Changes in the tension in response to agonist stimulation were measured isometrically, and recorded on a recorder. Acetylcholine, 10⁻⁵M, was administered to induce contraction of the ileum specimen; after the contraction maximized, the bath was washed three times to purge out the acetylcholine. This operation was repeated at 10-minute intervals, and after the induced contraction stabilized two consecutive times, each test substance was administered. By comparing the contractile forces produced by acetylcholine and the test substance, the effect of the test substance was evaluated. One specimen was investigated only at one concentration of the test substance. (1) For each of allyl isothiocyanate, cinnamic aldehyde, and acrolein, an investigation was made by single-dose administration at four concentrations: 10 μM, 30 μM, 100 μM, and 300 μM. As a result, constrictive action was observed at 100 μM or more for allyl isothiocyanate and cinnamic aldehyde, and at 10 μM or more for acrolein (Table 3). From the results above, it was shown that the TRPA1 activator induced gut contraction.

TABLE 3 Dose-dependent constrictive actions of allyl isothiocyanate, cinnamic aldehyde, and acrolein Magnus (guinea pig ileum) Action EC₅₀(μM) † Allyl Contraction 129.0 isothiocyanate Cinnamic aldehyde Contraction 88.3 Acrolein Contraction 70.3 († Calculated relative to constrictive reaction at 300 μM as 100%)

(2) Antagonization Experiments with TRPA1 Inhibitor (Ruthenium Red)

The inhibitory action of the TRPA1 receptor inhibitor Ruthenium Red (30 μM) on contraction upon stimulation with allyl isothiocyanate (300 μM) was investigated. For each of two different specimens from the same individual, either a vehicle or Ruthenium Red (30 μM) was applied for 15 minutes, after which contraction upon stimulation with allyl isothiocyanate (300 μM) was measured. As a result, in the Ruthenium Red-applied specimen, the contraction upon stimulation with allyl isothiocyanate was suppressed by about 84% compared to the vehicle-applied specimen. From the results above, it is suggested that allyl isothiocyanate may induce gut contraction via the TRPA1 receptor.

(3) Inhibition Experiments with Serotonin Receptor Antagonists

The inhibitory actions of various serotonin receptor antagonists on contraction upon stimulation with allyl isothiocyanate (300 μM) were investigated. The serotonin receptor antagonists used were pizotifen maleate (10 μM), a 5-HT1,2 receptor antagonist, ketanserin tartrate (0.1 μM), a 5-HT2 receptor antagonist, ramosetron hydrochloride (0.3 μM), a 5-HT3 receptor antagonist, and GR113808 (0.3 μM), a 5-HT4 receptor antagonist. In different specimens from the same individual, a vehicle or each serotonin antagonist was applied for 15 minutes, after which contraction upon stimulation with allyl isothiocyanate (300 μM) was measured. As a result, in the pizotifen maleate-applied specimen and ramosetron hydrochloride-applied specimen, contraction upon stimulation with allyl isothiocyanate was suppressed by about 44% and about 74%, respectively, compared to the vehicle-applied specimen. From the results above, it was shown that serotonin was released upon stimulation with allyl isothiocyanate to induce contraction via serotonin receptors such as 5-HT1 receptor and 5-HT3 receptor.

The inhibitory actions of various serotonin receptor antagonists on contraction upon stimulation with acrolein (300 μM) were investigated in the same manner. As a result, in the pizotifen maleate-applied specimen and ramosetron hydrochloride-applied specimen, contraction upon stimulation with allyl isothiocyanate was suppressed by about 74% and about 84%, respectively, compared to the vehicle-applied specimen. From the results above, it was shown that serotonin was released upon stimulation with acrolein to induce contraction via serotonin receptors such as 5-HT1 receptor and 5-HT3 receptor.

From the results obtained in the Examples above, it was demonstrated that TRPA1 is highly expressed in the digestive tract, particularly in the gut EC cells. Furthermore, as a result of an extensive investigation using a TRPA1 activator and inhibitor obtained by performing compound screening, it was demonstrated that TRPA1 activation causes serotonin release from gut EC cells, and causes gut contraction via the released serotonin. Next, in the Examples below, a test was performed to determine whether the TRPA1 activator has the action of accentuating digestive tract movement in vivo.

Example 20 Measurements of the Action of Accentuating Dog Digestive Tract Movement

Measurements of digestive tract movement were performed by the strain gauge force transducer method. Dogs (beagle dogs, male, 11-13 kg), fasted for 24 hours, under pentobarbital sodium anesthesia, had a strain gauge force transducer (F-12IS, Star Medical, Inc., Tokyo) sutured to a total of four sites, i.e., a portion 5 cm from the pylorus toward the mouth (gastric vestibule), a portion 20 cm from the Treiz ligament toward the anus (jejunum), a portion 10 cm from the ileocecal opening toward the anus (proximal colon), and a portion 10 cm from the anus toward the mouth (distal colon), in a way that allowed examination of contraction along the orbicular muscle. After recovery for 1 week or more postoperatively, experiments were preformed. Measurements of digestive tract movement were performed using a telemeter system (DAT-80RA, Star Medical, Inc.). Allyl isothiocyanate was administered orally about 20 minutes after phase-III-like digestive tract movement in the stomach was measured after regular expression of IMC (Inter digestive migrating motor complex) was confirmed by a measurement of digestive tract movement in animals previously fasted for 17 hours or more. As a result, as shown in FIG. 8-1, allyl isothiocyanate (1, 10 mg/kg) induced colon GMC (Giant migrating contraction) within 10 minutes after administration; it is suggested that allyl isothiocyanate, a TRPA1 activator, might accentuate digestive tract movement to induce defecation. On the other hand, as shown in FIG. 8-2, in the vehicle group, induction of GMC was not observed.

Because 5-HT is known to accentuate water secretion from the digestive tract, if a TRPA1 activator secretes 5-HT via gut EC cells, it is expected to accentuate water secretion from the digestive tract. Hence, an actual test was performed to determine whether or not a TRPA1 activator has the action of accentuating water secretion from the digestive tract.

Example 21 Measurement of Mouse Gut Water Secretion Secretion Accentuating Action

Mice (ddy, male, 35-42 g, SLC), fasted overnight, were anesthetized with pentobarbital (50 mg/kg i.p.) and laparotomized, and ileum tissue about 2 cm in the vicinity of the cecum was ligated with a thread at both ends to prepare an ileum loop. 100 μL of saline or allyl isothiocyanate, a TRPA1 activator (10, 100, 1,000 μg), was administered into the loop. After the administration, the gut was returned to the original position, and the abdominal muscle and the skin were sutured. Six hours after the treatment, each mouse was killed by cervical dislocation, after which the ileum loop was extirpated, and the content was weighed. As a result, allyl isothiocyanate dose-dependently accentuated water secretion from the gut, with significant water secretion accentuating action observed in the 1,000 μg dose group (FIG. 9).

Example 22 Evaluation of Allyl Isothiocyanate Using a Mouse Constipation Model

Because loperamide, a μ opioid receptor agonist, induces convulsive contraction in the gut and causes a delay of gut transportation, this experimental system is thought to be an experimental model of constipation type irritable bowel syndrome. Hence, an investigation was performed to determine whether or not allyl isothiocyanate, a TRPA1 activator, is effective in this constipation model.

Mice (ddY, male, 5-week-old, SLC) were fasted from afternoon of the day before the experiment; on the day of the experiment, the mice were acclimated to the measurement cage for 1 hour or more, after which loperamide, 0.3 mg/kg, was administered subcutaneously. After 30 minutes, allyl isothiocyanate, a TRPA1 agonist, 0.01 to 1 mg/kg, was administered orally, just after which each mouse was anesthetized with ether, and had glass beads 3 mm in diameter inserted to a position 2 cm from the anus. The mouse was returned to the measurement cage, and time from awakening to discharge of the glass beads was measured. As a result, as shown in FIG. 10, a delay in bead discharge time was observed in the loperamide-dosed group (vehicle group), compared to the loperamide-non-dosed group (control group). Allyl isothiocyanate, a TRPA1 agonist, dose-dependently ameliorated the delay of bead discharge time by loperamide. From the results above, it is suggested that the TRPA1 activator might be effective against constipation type irritable bowel syndrome.

While the present invention has been described along with specific embodiments, modifications and improvements obvious to those of ordinary skill in the art are encompassed in the scope of the present invention.

This application is based on patent application No. 2006-275837 filed in Japan (filing date: Oct. 6, 2006), and the contents disclosed therein are hereby entirely incorporated by reference. In addition, the contents disclosed in any publication cited herein, including patents and patent applications, are hereby incorporated in their entireties by reference, to the extent that they have been disclosed herein.

Claims

1. A screening method for a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, comprising evaluating a test substance to determine whether or not the test substance is capable of regulating the expression or channel activity of TRPA1.

2. A screening method for a prophylactic and/or therapeutic drug for digestive organ diseases, comprising evaluating a test substance to determine whether or not the test substance is capable of regulating the expression or channel activity of TRPA1.

3. The screening method of claim 1, comprising the following steps (a) to (c):

(a) a step for bringing a test substance into contact with mammalian cells that are expressing TRPA1;

(b) a step for analyzing the expression or channel activity of TRPA1; and

(c) a step for selecting a substance capable of regulating the expression or channel activity of TRPA1.

4. The screening method of claim 3, wherein the mammalian cells that are expressing TRPA1 are chromaffin cells, pancreatic β cells or cells transformed with a TRPA1 expression vector.

5. The screening method of claim 1, wherein the screening method is performed using a TRPA1 activator or a TRPA1 inhibitor.

6. The screening method of claim 1, wherein the regulation of the expression or channel activity of TRPA1 is promotion of the expression or channel activity of TRPA1.

7. The screening method of claim 1, wherein the regulation of the expression or channel activity of TRPA1 is suppression of the expression or channel activity of TRPA1.

8. The screening method of claim 3, which is a method of screening for a prophylactic or therapeutic drug for constipation type irritable bowel syndrome, functional dyspepsia or constipation by selecting a substance capable of promoting the expression or channel activity of TRPA1.

9. The screening method of claim 3, which is a method of screening for a prophylactic or therapeutic drug for diarrhea type irritable bowel syndrome, diarrhea or vomiting by selecting a substance capable of suppressing the expression or channel activity of TRPA1.

10. A screening tool for a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, comprising cells transformed with a TRPA1 expression vector.

11. A screening tool for a prophylactic and/or therapeutic drug for a digestive organ disease, comprising cells transformed with a TRPA1 expression vector.

12. The screening tool of claim 11, wherein the digestive organ disease is irritable bowel syndrome, functional dyspepsia, constipation, diarrhea or vomiting.

13. A prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, comprising a substance capable of regulating the expression or channel activity of TRPA1.

14. A prophylactic and/or therapeutic drug for a digestive organ disease, comprising a substance capable of regulating the expression or channel activity of TRPA1.

15. A method of producing a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, comprising screening for a substance capable of regulating the expression or channel activity of TRPA1, and preparing the substance obtained by the screening as a pharmaceutical preparation.

16. The production method of claim 15, comprising the following steps (a) to (d):

(a) a step for bringing a test substance into contact with mammalian cells that are expressing TRPA1;

(b) a step for analyzing the expression or channel activity of TRPA1;

(c) a step for selecting a substance capable of regulating the expression or channel activity of TRPA1; and

(d) a step for preparing the substance obtained in the step (c) as a pharmaceutical preparation.

17. A prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, comprising a substance that is obtained by the screening method of claim 1.

18. A prophylactic and/or therapeutic method for diseases associated with 5-HT production/secretion abnormalities, comprising administering a substance that is obtained by the screening method of claim 1 to a patient in need of prevention and/or treatment.

19. A method of producing a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities comprising employing a substance that is obtained by the screening method of claim 1.

20. A screening method for a substance that exhibits a specified pharmacological effect, and that does not have the capability of regulating 5-HT release, comprising evaluating a test substance to determine whether or not the test substance that exhibits a specified pharmacological effect is capable of regulating the expression or channel activity of TRPA1.

21. The screening method of claim 20, comprising the following steps (a) to (c):

(a) a step for bringing a test substance that exhibits a specified pharmacological effect into contact with mammalian cells that are expressing TRPA1,

(b) a step for analyzing the expression or channel activity of TRPA1; and

(c) a step for selecting a substance that exhibits a specified pharmacological effect, and that does not regulate the expression or channel activity of TRPA1.

22. A method of producing a pharmaceutical, comprising screening for a substance that exhibits a specified pharmacological effect, and that does not regulate the expression or channel activity of TRPA1, and preparing the substance obtained by the screening as a pharmaceutical preparation.

23. A screening tool for a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities comprising TRPA1.

24. A screening tool for a prophylactic and/or therapeutic drug for a digestive organ disease comprising TRPA 1.

25. The screening method of claim 2, comprising the following steps (a) to (c):

(a) a step for bringing a test substance into contact with mammalian cells that are expressing TRPA1;

(b) a step for analyzing the expression or channel activity of TRPA1; and

(c) a step for selecting a substance capable of regulating the expression or channel activity of TRPA1.

26. The screening method of claim 25, wherein the mammalian cells that are expressing TRPA1 are chromaffin cells, pancreatic B cells or cells transformed with a TRPA1 expression vector.

27. The screening method of claim 2, wherein the screening method is performed using a TRPA1 activator or a TRPA1 inhibitor.

28. The screening method of claim 2, wherein the regulation of the expression or channel activity of TRPA1 is promotion of the expression or channel activity of TRPA1.

29. The screening method of claim 2, wherein the regulation of the expression or channel activity of TRPA1 is suppression of the expression or channel activity of TRPA1.

30. The screening method of claim 25, which is a method of screening for a prophylactic or therapeutic drug for constipation type irritable bowel syndrome, functional dyspepsia or constipation by selecting a substance capable of promoting the expression or channel activity of TRPA1.

31. The screening method of claim 25, which is a method of screening for a prophylactic or therapeutic drug for diarrhea type irritable bowel syndrome, diarrhea or vomiting by selecting a substance capable of suppressing the expression or channel activity of TRPA1.

32. A prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities, comprising a substance that is obtained by the screening method of claim 2.

33. A prophylactic and/or therapeutic method for diseases associated with 5-HT production/secretion abnormalities, comprising administering a substance that is obtained by the screening method of claim 2 to a patient in need of prevention and/or treatment.

34. A method of producing a prophylactic and/or therapeutic drug for diseases associated with 5-HT production/secretion abnormalities comprising employing a substance that is obtained by the screening method of claim 2.